The main thrust of this research proposal is to demonstrate the manner in which the redox state of nicotinamide adenine dinucleotide (NAD ion), as reflected by the ratio of free oxidized to free reduced NAD ion, viz., NAD ion/NADH, is operative in the assessment of the metabolic status of cells, with particular attention to those changes evoked by hypoxia in erythrocytes. The research goals include: 1) confirmation (or refutation) of Krebs' observation that the redox state of the NAD ion-NADH couple of the LDH-catalyzed system is equal to that determined by the combined GAPDH-PGK-catalyzed reaction; 2) determination of the role of the adenine nucleotides in the control of the nicotinamide-adenine dinucleotide system in the erythrocyte; 3) determination of the role of the state of oxygenation of hemoglobin in the control of the nicotinamide-adenine nucleotide system in normal and sickle erythrocytes, with particular attention to the adenine nucleotide system as manifest by the phosphorylation ratio, ATP/ADP.Pi. The proposed objectives will be accomplished by 1) substrate analysis of each of the NAD ion-dependent dehydrogenase-catalyzed reactions in the glycolytic pathway in the erythrocyte; demonstration of the pH characteristics of each dehydrogenase catalyzed reaction; calculation of the value of the NAD ion/NADH ratio; 2) with the information provided by the preceding, the phosphorylation ratio and the NAD ion/NADH ratio will be determined as a function of hemoglobin saturation in normal and sickle erythrocytes.